1. Field of the Invention
This invention pertains to the field of diagnostic methods and kits for detecting Clostridium difficile. The methods and kits provide rapid, sensitive, and accurate assays for the presence of toxigenic strains of C. difficile in an biological sample.
2. Background
Clostridium difficile, an anaerobic organism, is the major causative agent of pseudomembranous colitis (PMC) in humans. PMC is characterized by diarrhea, a severe inflammation of the colonic mucosa, and formation of pseudomembranes that are composed of fibrin, mucus, necrotic epithelial cells, and leukocytes. The pseudomembrane can form a sheath over the entire colonic mucosa. In addition to causing PMC, C. difficile is believed to play a role in other less severe gastrointestinal illnesses; the organism is estimated to cause approximately 25% of reported cases of antibiotic-associated diarrhea. Brettle and Wallace (1984) J. Infect. 8: 123-128; Gilligan et al. (1981) J. Clin. Microbiol. 14: 26-31. Diarrhea affects approximately 25 million persons annually in the US alone, and causes almost 11,000 deaths. Peterson and Kelly (1993) Lab. Diagnosis Infect. Dis. 7: 277-292. C. difficile-caused diseases are not limited to gastrointestinal illnesses, as the organism can cause abscesses, wound infections, osteomyelitis, urogenital tract infections, septicemia, peritonitis, and pleuritis. Lyerly et al. (1988) Clin. Microbiol. Rev. 1: 1-18; Hafiz et al. (1975) Lancet 1: 420-421; Levett (1986) J. Infect. 12: 253-263; Saginur et al. (1983) J. Infect. Dis. 147: 1105. Antibiotics can predispose a host animal to PMC and other C. difficile-related illnesses, as the disturbance of the normal bacterial flora by the antibiotic disrupts the major barrier against colonization by pathogens, rendering the host animal susceptible to colonization by pathogens such as C. difficile. Hospitals and chronic care facilities are significant sources of C difficile infection, with one study finding that 21% of patients acquired C. difficile infection during hospitalization. McFarland et al. (1989) N. Engl. J. Med. 320: 204.
The high frequency of C. difficile infection, coupled with the likelihood of a poor clinical outcome for cases that are not treated promptly, makes clear the need for rapid and accurate tests to detect C. difficile infection, determine whether any C. difficile present is toxigenic, and evaluate the effectiveness of treatment. Previously available methods for detecting C. difficile are much less than optimal for effective diagnosis and treatment of infection. One previously known method for detecting C. difficile infection is culture on agar media. The efficacy of this method is hampered by the significant variation in results that are obtained using different media, and the high rate of false positives (10-29%). Peterson and Kelly, supra. An additional disadvantage of this method is the lengthy culture period required before visible C. difficile colonies are discernable. A commercially available assay for C. difficile involves latex agglutination of an antigen that was eventually identified as C. difficile glutamate dehydrogenase. Lyerly et al. (1991) J. Clin. Microbiol. 29: 2639; Lyerly et al. (1986) J. Clin. Microbiol. 23: 622. However, this assay suffers from widely varying and insufficient sensitivity and specificity, with sensitivity ranging from 68% to 93% and specificity between 80% and 95%. Peterson and Kelly, supra. Moreover, previously available glutamate dehydrogenase assays were not thought to be useful for detection of toxigenic strains of C. difficile because glutamate dehydrogenase is produced by non-toxigenic strains of C. difficile, as well as toxigenic strains.
Non-toxigenic strains of C. difficile are generally considered clinically insignificant, while toxigenic strains can be lethal. Although distinguishing between toxigenic and non-toxigenic C. difficile strains is thus of great importance, previously known assays that were used in attempts to accomplish this goal were ineffective. One commonly used diagnostic method for detecting the presence of toxigenic C. difficile involves determining whether C. difficile is cytotoxic to susceptible cell lines. This cytotoxicity is the result of either or both of the two toxins produced by C. difficile, an enterotoxin designated toxin A and a cytotoxin designated toxin B, both of which are believed to be involved in the pathogenesis of PMC. The cytotoxicity assay has significant drawbacks for clinical use however, including the need to maintain tissue culture lines and the relatively low sensitivity of the assay. For example, Peterson and Kelly, supra., found that the sensitivity of cytotoxin detection alone ranged from 67% to 100%, and other researchers found sensitivity to be as low as 71%. Demlee et al. (1985) J. Clin. Microbiol. 21: 323. Immunoassays for toxins A and/or B have also been used to detect C. difficile in samples, but these methods suffer from low sensitivity (63% to 88%). Peterson and Kelly, supra.
Thus, a need exists for assays to detect the presence of C. difficile in a sample that are rapid, sensitive, specific, and cost-effective. Assays to determine whether an infecting C. difficile strain is toxigenic are also needed. The present invention fulfills these and other needs.
The present invention provides methods, compositions, and kits for the rapid detection of C. difficile in a test sample, in particular for detection of toxigenic C. difficile strains.
In a first embodiment, the methods involve detecting C. difficile toxin A or toxin B, and also detecting C. difficile glutamate dehydrogenase. The assay means for detecting the C. difficile antigens can be, for example, immunoassays. Sandwich assays provide a convenient, sensitive method for performing the methods. In one embodiment, the assay involves detection of C. difficile toxin A using a sandwich assay that employs an anchor moiety that is immobilized on a solid support and specifically binds to at least a first epitope of C. difficile toxin A, and one or more detection moieties, each of which is conjugated to a detectable label and specifically binds to at least a second epitope of C. difficile toxin A. The assay for detecting C. difficile glutamate dehydrogenase can also involve a sandwich assay in which glutamate dehydrogenase is bound to an immobilized anchor moiety that specifically binds to at least a first epitope of C. difficile glutamate dehydrogenase, after which bound glutamate dehydrogenase is detected using one or more detection moieties, each of which is conjugated to a detectable label and specifically binds to at least a second epitope of C. difficile glutamate dehydrogenase. The anchor moiety that specifically binds C. difficile toxin A and the anchor moiety that specifically binds C. difficile glutamate dehydrogenase can be immobilized in separate zones on a single solid support, on separate supports, or can both be present in a single zone.
The invention also provides devices for detecting the presence of toxigenic strains of C. difficile in a test sample. The devices include a porous member having an upper and a lower surface, being positioned in the device such that the test sample may be applied to the upper surface, wherein a plurality of anchor moieties that are capable of specifically binding to C. difficile toxin A are immobilized in a first zone of the porous member, and a plurality of anchor moieties that are capable of specifically binding to C. difficile glutamate dehydrogenase are immobilized in a second zone of the porous member. The devices also include a non-absorbent member having a textured surface with channels capable of forming a network of capillary channels when placed in communication beneath or around the porous member, said-capillary network being substantially parallel to the lower surface of the porous member. The test sample, alone or in combination with other fluids, when applied to the upper surface is drawn through the porous member to the capillary network formed between the porous member and the non-absorbent member when substantially all the void volume of the porous member is filled with the test sample and when contact is made between the porous member and the non-absorbent member.
Kits for performing these assays for C. difficile are also provided by the present invention. The kits can include a container that includes some or all of the reagents and methods for carrying out the assays. For example, a kit can include a solid support upon which is immobilized an anchor moiety that specifically binds to C. difficile toxin A and an anchor moiety that specifically binds to C. difficile glutamate dehydrogenase. Also included in the kits can be a detection moiety that specifically binds to C. difficile toxin A, a detection moiety that specifically binds to C. difficile glutamate dehydrogenase, and reagents useful for detecting the presence of two different detectable labels present on each of the detection moieties. Written instructions as to how to use the kit to assay for the presence of C. difficile toxin A and glutamate dehydrogenase in a test sample can also be provided in the kits. The kits can also include, as controls, C. difficile toxin A and/or glutamate dehydrogenase; the C. difficile antigen can be complexed with the anchor moiety in a control zone of the solid support.
Another embodiment of the invention provides methods, compositions, and kits for detecting the presence of toxigenic C. difficile strains in a test sample by performing a highly sensitive assay for C. difficile glutamate dehydrogenase. These methods are capable of detecting the presence of toxigenic C. difficile in samples that would have tested negative for toxigenic C. difficile using previously available assays. One method of performing the highly sensitive assays is to contact the sample with a solid support to which is bound an anchor moiety that is specific for at least a first epitope of C. difficile glutamate dehydrogenase for a time sufficient for some or all of the glutamate dehydrogenase to bind to the anchor moiety. The bound C. difficile glutamate dehydrogenase is then contacted with one or more detection moieties, each of which includes a detectable label and a binding moiety that is capable of specifically binding to at least a second epitope of C. difficile glutamate dehydrogenase. The presence of bound detectable label is indicative of toxigenic C. difficile in the sample.
Another embodiment of the invention provides methods for accurately determining whether a test sample contains a toxigenic strain of C. difficile by testing for the presence of C. difficile glutamate dehydrogenase in the test sample, and, if C. difficile glutamate dehydrogenase is present in the test sample, testing for the presence of C. difficile toxin A or toxin B in the sample. Preferably, the toxin A or toxin B assay has a sensitivity of at least about 2 ng toxin per ml. Any of several assays can be used to detect the C. difficile antigens. For example, the testing for the presence of C. difficile toxin A or toxin B can involve amplification of a nucleic acid that encodes toxin A or toxin B, or a portion of said nucleic acid, by polymerase chain reaction and detecting the presence of the amplified nucleic acid. Toxin B can be detected by means of a cytotoxicity assay. Additional high sensitivity assays for use in these methods are described herein.
In another embodiment, the invention provides methods for concentrating C. difficile toxin A from a test sample by using magnetic particles. These methods involve adding to the test sample a toxin A binding moiety that specifically binds to C. difficile toxin A to form a binding moiety-toxin A complex, and adding to the test sample a magnetic bead to which is attached a capture moiety that specifically binds to the toxin A binding moiety to form a magnetic bead-binding moiety-toxin A complex. The magnetic bead-binding moiety-toxin A complex is separated from the test sample, after which the binding moiety-toxin A complex is dissociated from the magnetic bead. The magnetic bead can then be separated from the solution containing the binding moiety-toxin A complex and the presence of the binding moiety-toxin A complex is then detected. In a preferred embodiment of the magnetic bead-based toxin A concentration method, the binding of the capture moiety to the toxin A binding moiety is reversible under relatively mild conditions. Reversible binding can be achieved by linking to the toxin A binding moiety a molecular tag to which the capture moiety binds. The molecular tag and the corresponding capture moiety are chosen so as to dissociate under conditions that do not disrupt the ability of the toxin A binding moiety to bind to the toxin A. If the dissociation conditions also cause the toxin A binding moiety and toxin A to dissociate, then following separation of the magnetic beads, the concentrated toxin A solution can be modified so that the toxin A binding moiety and the toxin A can immediately reassociate. For example, if the pH of the solution was increased in order to effect dissociation of the magnetic beads from the toxin A binding moiety, after separation of the beads the solution can be modified by neutralization.
Another embodiment of the invention provides methods of detecting toxin A that has been concentrated using the methods described herein. The detection method can involve applying the solution containing a toxin A binding moiety-toxin A complex prepared as above to a solid support upon which is immobilized an anchor moiety that is capable of specifically binding to an epitope of C. difficile toxin A that is different than the toxin A epitope to which the toxin A binding moiety binds. A detection moiety that is capable of specifically binding to the toxin A binding moiety, or to a hapten attached to the binding moiety, is used to detect the presence of immobilized toxin A and associated toxin A binding moiety.
Kits provided by the invention can include a toxin A binding moiety that specifically binds to C. difficile toxin A, a magnetic bead to which is attached a capture binding moiety that specifically binds to the toxin A binding moiety, a solid support upon which is immobilized an anchor binding moiety that specifically binds to at least one epitope of C. difficile toxin A that is different than the toxin A epitope to which the toxin A binding moiety binds, and a detection moiety that is conjugated to a detectable label and specifically binds to a hapten present on the toxin A binding moiety.